BIOORGANIC & MEDICINAL CHEMISTRY
·
2021
·
IF 3.641
Yaping Cheng,Jingyuan Wu,Ying Han,Jingyao Xu,Yifan Da,Qian Zhao,Guoying Guo,Yani Zhou,Yimin Chen,Jinghong Liu,Huayao Chen,Xianxing Jiang,Xiaoqing Cai
Covalent target modulation with small molecules has been emerging as a promising strategy for drug discovery. However, covalent inhibitory antibody remains unexplored due to the lack of efficient strategies to engineer antibody with desired bioactivity. Herein, we developed an intracellular selection method to generate covalent inhibitory antibody against human rhinovirus 14 (HRV14) 3C protease through unnatural amino acid mutagenesis along the heavy chain complementarity-determining region 3 (CDR-H3). A library of antibody mutants was thus constructed and screened in vivo through co-expression with the target protease. Using this screening strategy, six covalent antibodies with proximity-enabled bioactivity were identified, which were shown to covalently target HRV14-3C protease with high inhibitory potency and exquisite selectivity . Compared to structure-based rational design, this library-based screening method provides a simple and efficient way for the discovery and engineering of covalent antibody for enzyme inhibition.
研究领域:
药物发现
·
蛋白质工程
·
病毒学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2022
·Vol. 106
·期 23
·7805-7817
·
IF 5.56
Fu Linglong,Sun Mengning,Wen Weizhang,Dong Na,Li Defa
Abstract Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1 403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein ( pal ) gene. Ulp1 403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δ pal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1 403-621 approximately four fold. Moreover, extracellular Ulp1 403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus . The specific activity of extracellular Ulp1 403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 10 6 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. Key points
• First report of extracellular production of Ulp1
403-621
in leaky Escherichia coli BL21(DE3) strain.
• One percent glycine addition into cultivation medium increased the extracellular production of Ulp1
403-621
approximately four fold.
• The specific activity of extracellular Ulp1
403-621
produced in this study reached 2.0 × 10
6
U/L.
研究领域:
生物技术
·
微生物学
·
蛋白质工程
·
药学
✓ 已验证
DOI
PubMed
高影响因子
INTERNATIONAL IMMUNOPHARMACOLOGY
·
2024
·
IF 4.8
Shu-Zhen Dai,Ri-Hong Wu,Hengyu Chen,Ming-Hui Chen,Weijing Xie,Wu-Ping Zheng,Guang-Hong Tan,Feng-Ying Huang
Background The process of NETosis is observed in a range of inflammatory conditions. Progesterone (P4) has been shown to alleviate inflammation caused by viral infections such as influenza and SARS-CoV-2. However, the precise molecular mechanisms responsible for this effect are not yet fully understood. Therefore, the present investigation aims to explore whether P4 can exert its anti-inflammatory properties by inhibiting NETosis and the related molecular pathways. Methods Airway inflammation caused by rhinovirus serotype-1b (RV-1b) was induced in male BALB/c mice. The inflammation was assessed through histological examination and calculation of inflammatory cells present in the bronchoalveolar lavage fluid. Flow cytometry was used to analyze the inflammatory cells and NETotic neutrophils. Western blotting analysis was conducted to detect proteins associated with NETosis, inflammasome activation, and signaling. Furthermore, confocal microscopy was utilized to observe neutrophil extracellular trap (NET) structures in vivo tissues and in vitro neutrophils, neutrophil infiltration, and inflammasome formation. Results The administration of P4 proved to be an effective treatment for reducing airway inflammation and the production of NETs caused by RV-1b infection. The infection triggered the activation of NLRP3 inflammasomes in neutrophils, which led to the maturation of IL-1β and subsequent activation of both the NF-κB and p38 signaling pathways. The activation of NF-κB signaling resulted in the secretion of downstream chemokines CCL3 and IL-6, which led to an increase in neutrophil infiltration into the lung airways. Moreover, the activation of p38 signaling led to the generation of reactive oxygen species, resulting in NETosis. However, the administration of P4 inhibited the activation of the NLRP3 inflammasome, which subsequently led to the deactivation of both the IL-1β-NF-κB and IL-1β-p38 axes. As a result, there was a reduction in neutrophil infiltration and NETosis. Furthermore, TGF-β-activated kinase 1 (TAK1) was identified as an intermediary enzyme. P4 inhibits both the NF-κB and IL-1β-p38 pathways by suppressing the activity of TAK1. Conclusion The capacity of P4 to mitigate rhinovirus-induced airway inflammation is attributed to its ability to impede the infiltration of neutrophils and NETosis. As inflammation mediated by NETosis is widespread in diverse disorders, our findings propose that P4 could potentially function as a universal therapeutic agent in the management of such ailments.
研究领域:
免疫学
·
呼吸病学
·
病毒学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ADVANCED MATERIALS
·
2024
·
IF 27.4
Changhao Dai,Huiwen Xiong,Rui He,Chenxin Zhu,Pintao Li,Mingquan Guo,Jian Gou,Miaomiao Mei,Derong Kong,Qiang Li,Andrew Thye Shen Wee,Xueen Fang,Jilie Kong,Yunqi Liu,Dacheng Wei
On-site diagnostic tests that accurately identify disease biomarkers lay the foundation for self-healthcare applications. However, these tests routinely rely on single-mode signals and suffer from insufficient accuracy, especially for multiplexed point-of-care tests (POCTs) within a few minutes. Here, this work develops a dual-mode multiclassification diagnostic platform that integrates an electrochemiluminescence sensor and a field-effect transistor sensor in a microfluidic chip. The microfluidic channel guides the testing samples to flow across electro-optical sensor units, which produce dual-mode readouts by detecting infectious biomarkers of tuberculosis (TB), human rhinovirus (HRV), and group B streptococcus (GBS). Then, machine-learning classifiers generate three-dimensional (3D) hyperplanes to diagnose different diseases. Dual-mode readouts derived from distinct mechanisms enhance the anti-interference ability physically, and machine-learning-aided diagnosis in high-dimensional space reduces the occasional inaccuracy mathematically. Clinical validation studies with 501 unprocessed samples indicate that the platform has an accuracy approaching 99%, higher than the 77%–93% accuracy of rapid point-of-care testing technologies at 100% statistical power (>150 clinical tests). Moreover, the diagnosis time is 5 min without a trade-off of accuracy. This work solves the occasional inaccuracy issue of rapid on-site diagnosis, endowing POCT systems with the same accuracy as laboratory tests and holding unique prospects for complicated scenes of personalized healthcare.
研究领域:
生物医学工程
·
即时诊断
·
微流控技术
·
机器学习
·
生物标志物分析
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）